In scanning microscopy, a sample is illuminated with a light beam in order to observe the detection light emitted, as reflected or fluorescent light, from the sample. The focus of an illuminating light beam is moved in a sample plane by means of a controllable beam deflection device, generally by tilting two mirrors, the deflection axes usually being perpendicular to one another so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the detection light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the present mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The detection light coming from the specimen, which for example can be fluorescent or reflected light, travels back through the beam deflection device to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located. This detection configuration is called a “descan” configuration. Detection light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which results, by sequential scanning of the specimen with the focus of the illuminating light beam, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers.
In confocal scanning microscopy, a detection pinhole can be dispensed with in the case of two-photon (or multi-photon) excitation, since the excitation probability depends on the square of the photon density and thus on the square of the illuminating light intensity, which of course is much greater at the focus than in the adjacent regions. The fluorescent light being detected therefore very probably originates almost exclusively from the focus region, which renders superfluous any further differentiation, using a pinhole arrangement, between fluorescent photons from the focus region and fluorescent photons from the adjacent regions.
A non-descan configuration, in which the detection light does not travel to the detector via the beam deflection device (descan configuration) and the beam splitter which couples in the illuminating light, but rather is conveyed directly to a non-descan detector, is of interest especially in view of the already low fluorescent photon yield with two-photon excitation, since less light is generally lost along this detection light path. The non-descan detector can be arranged on the condenser side, i.e. on the side of the sample opposite from the objective. It is also possible to separate detection light proceeding from the sample out of the illuminating beam path using a dichroic beam splitter on the objective side, and convey it to a non-descan detector. Arrangements of this kind are known, for example, from the publication of David W. Piston et al., “Two-photon excitation fluorescence imaging of three-dimensional calcium-ion activity,” Applied Optics, Vol. 33, No. 4, February 1996; and from Piston et al., “Time-Resolved Fluorescence Imaging and Background Rejection by Two-Photon Excitation in Laser Scanning Microscopy,” SPIE Vol. 1640. Both of these publications are hereby incorporated by reference herein.
In known scanning microscopes, especially in the case of samples whose detection light has a very low power level, the theoretically possible image contrast is not achieved despite the most careful setup. This is attributable in particular to the fact that the detectors, especially the non-descan detectors used in a non-descan configuration, are continuously exposed to incident light, especially ambient light. This contributes to accelerated aging of the detectors and causes, in particular, undesired dark currents and background currents and poor noise behavior, and the detectors' sensitivity is reduced.
Premature aging or even instantaneous damage to or destruction of sensitive detectors is often caused by excessively high detection light power levels.